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The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC-precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.

Original publication

DOI

10.1021/acs.biochem.8b00026

Type

Journal article

Journal

Biochemistry

Publication Date

13/03/2018

Volume

57

Pages

1663 - 1671

Keywords

Chromatography, Reverse-Phase, Detergents, Diosgenin, Escherichia coli, Escherichia coli Proteins, Mass Spectrometry, Membrane Transport Proteins, Native Polyacrylamide Gel Electrophoresis, Surface Plasmon Resonance